Methods to fix and detect nucleic acids

ABSTRACT

In one aspect, the invention relates to a method for fixing a short nucleic acid in a biological sample. In another aspect, the invention relates to a method for detecting a target short nucleic acid in a biological sample. The method includes contacting the biological sample with an aldehyde-containing fixative, and subsequently contacting the sample with a water-soluble carbodiimide. In a further aspect, the invention relates to a kit for fixing a short nucleic acid in a biological sample. The kit includes a support substrate for holding the sample; an aldehyde-containing fixative; and a water-soluble carbodiimide.

This application asserts priority to U.S. Provisional Application Ser.No. 61/159,288 filed on Mar. 11, 2009, which is hereby incorporated byreference in its entirety.

The invention was made with U.S. Government support under contractnumber GM073047, EY18082-01A2 and MH080442. Accordingly, the U.S.Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Association studies are rapidly linking miRNAs with cancer andneurological disorders. miRNAs have specific expression and function inspecialized cell types, emphasizing the need to definecell-type-specific miRNA expression patterns. The most common method forvisualizing gene expression in specific cell types is in situhybridization (ISH).

However, conventional ISH methods permit the release and diffusion ofsmall nucleic acids, such as miRNA, from tissue. Therefore, thereremains a need for an improved method to fix and detect small nucleicacids in a tissue sample.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for fixing a shortnucleic acid in a biological sample. The method includes contacting thebiological sample with an aldehyde-containing fixative, and subsequentlycontacting the sample with a water-soluble carbodiimide.

In another aspect, the invention relates to a method for detecting atarget short nucleic acid in a biological sample. The method includescontacting the biological sample with an aldehyde-containing fixative;subsequently contacting the sample with a water-soluble carbodiimide toproduce a crosslinked short nucleic acid; contacting the cross-linkedmiRNA with a probe, said probe being complementary to all or a part of aregion of interest of the short nucleic acid, thereby producing ahybridized short nucleic acid; and detecting the hybridized shortnucleic acid as the target short nucleic acid.

In a further aspect, the invention relates to a kit for fixing a shortnucleic acid in a biological sample. The kit includes a supportsubstrate for holding the sample; an aldehyde-containing fixative; and awater-soluble carbodiimide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: miRNAs retained in formaldehyde+EDC-fixed tissues. (a) Northernblotting analysis shows escape of miR-124 from formaldehyde-fixedtissues into ISH buffer after 24 h incubation at temperatures of 40° C.and higher. (b) Time course of hybridization at 50° C. shows miR-124accumulates in ISH buffer after 1 h. (c) Quantification of Northernblots show approx. 50% of miR-124 were present in the hybridizationbuffer after overnight incubation, P<0.05, n=3. (d) At 4° C., miR-124was not detectable in the hybridization buffer, which would suggest thatRNA-protein crosslinks were intact at lower temperatures. (e) Samplesfixed with formaldehyde+EDC show negligible amounts of miR-124 in ISHbuffer at pH 7.0 and 8.0.

FIG. 2: Visualization of miRNAs expressed at different levels in themouse brain. (a) Low magnification images of nervous system specificmiR-124 shows broad expression in neurons. (b-c) High magnificationimages of miR-124 demonstrate ubiquitous expression in the neurons ofthe cerebellum (top, orange), (b) cerebral cortex and hippocampus (c).(d) Higher magnification images show that miR-124 signals are notpresent in all cells, marked with arrows. (e) Fluorescence images ofmouse brain sections probed for highly expressed miR-9 (Cy3, red)localized in Purkinje cells of the cerebellum. (f,g) Image depictingmiR-410 (0 and miR-370 (g), which have an intermediate expression. (h,i)Images demonstrate robust signals for miRNAs differing by 3 nucleotides,miR-26b (h) and miR-26a (i) and these miRNAs were differentiallyexpressed. Cell nuclei were counterstained with4′,6-diamino-2-phenylindole dihydrochloride (DAPI, blue; bottom panel ofb-d; right panel of e-i). Scale bars, 500 μm (f-i).

FIG. 3: Formaldehyde+EDC-fixed sections show miRNA localized in thedendrites of neurons. (a) Low magnification fluorescent imagesdemonstrate a robust and broad distribution of miR-370 expression(white) with intense staining in the hippocampus (inset). (b,c) Highmagnification images show a single neuron in the CA1 region of thehippocampus for which miR-370 is localized both in the cell body and thedendrites (arrows) (c,d), represented by inset (a). miR-370 localizationextends 50 μm from the cell body (b), and 30 μM in other neurons (c,d).(e) The mature miRNA miR-9 expressed in dendrites of Purkinje cells inthe cerebellum, yet the miR-9*(0 was absent in dendrites. (g) Pyramidalcells in the hippocampus (arrows) show staining of miR-124 inhippocampus dendrites and in neurons located in the pons.

FIG. 4: Tissues fixed with formaldehyde-EDC substantially improves miRNAISH signal for less abundant miRNAs. (a) Low power fluorescentmicrograph images ISH medium expressed miR-130a in regions of theolfactory bulb and cerebral cortex from mouse brain tissues fixedformaldehyde+EDC (a, Cy3; left) or formaldehyde alone (b, Cy3; left).(c) Low power fluorescent micrograph images of ISH for miR-129-3p (c,Cy3; left) in the hippocampus and cerebral cortex from mouse braintissues fixed with formaldehyde and EDC (c, Cy3, left) or formaldehydealone. Nuclei were stained (DAPI, a-d, right). Scale bars 500 μm in a-d.

FIG. 5: Specific expression of miR-129-3p in the mouse brain andlocalization in dendrites. (a) Low power fluorescent micrograph imagesof the medium expressed miR-129-3p (Cy3; left) of the olfactory bulb andcerebral cortex and box in a represented in b, show higher magnificationmicrograph images with specific miR-129-3p expression in a single celllayer (b, Cy3, left). (c) Low power fluorescent micrograph images showmiR-129-3p (Cy3; left) expression in the cerebellum, box in c shown withhigher power magnification in panel d, show miR-129-3p (d, Cy3, left)expression in neuronal dendrites. Nuclei were stained (DAPI, right,a-d). Scale bars 500 μm in a, c; 200 μm in b; 100 μm in d.

FIG. 6: miRNA ISH signal intensity is EDC fixation pH dependent. (a) Lowpower fluorescent micrograph images of tissues fixed with formaldehydeand then EDC solution at pH 5.5 (a) and 6.0 (b) show reduced signal formiR-129-3p (Cy3), compared to ISH signal for miR-129-3p in tissues fixedwith EDC solution from pH 6.5 to 9.0 (d-h, Cy3). Scale bars 500 μm ina-h.

FIG. 7: EDC-based miRNA ISH is dependent on EDC concentration. (a-d) Lowpower fluorescent micrograph images of the medium expressed miR-130a(Cy3; left) fixed with formaldehyde-EDC at concentrations at or below 16mM EDC (a-d, Cy3, left). (e) Low power fluorescent micrograph imagesshow signal for miR-130a (e, Cy3; left) expression in the cortex with160 mM EDC. (f-h) Low power fluorescent micrograph images of the mediumexpressed miR-130a (Cy3; left) fixed with formaldehyde-EDC atconcentrations at or above 320 mM EDC (a-d, Cy3, left). Nuclei werestained (DAPI, blue; right, a-h). Scale bars 500 μm in a-h.

FIG. 8: Fixation with formaldehyde-cyanogen bromide (BrCN) enhancesmiRNA ISH detection in tissues. Fluorescent micrograph images of themouse olfactory bulb and cerebral cortex show expression of miR-130a(Cy3) in tissue sections fixed with formaldehyde (PFA)-EDC fixation (a,Cy3; left), formaldehyde-BrCN fixation (b, Cy3; left), and formaldehydefixation (c, Cy3; left). No probe control (d, Cy3; left). Nuclei werestained (DAPI, blue; right, a-d). Scale bars 500 μm in a-d.

FIG. 9: formaldehyde-BrCN tissue fixation is pH dependent. (a) Low powerfluorescent micrograph images of tissue sections of the mousehippocampus from tissues fixed with formaldehyde-BrCN in MES buffer showexpression of miR-129-3p (Cy3; a-h). Images of tissues fixed withformaldehyde and subsequent BrCN at pH solution at pH 5.5-8.5 showsignal for miR-129-3p (FIG. 7, a-g, Cy3) compared to ISH signal formiR-129-3p in tissues fixed with formaldehyde-BrCN solution at pH 9.0(h, Cy3), or tissues fixed with formaldehyde alone (i, Cy3). ISH signalat pH 7.0 showed the most intense staining (d), similar toformaldehyde-EDC (j, Cy3). Scale bars 500 μm in a-j.

FIG. 10: Fixation of tissue samples with formaldehyde-EDC-BrCN improvesmicroRNA detection in mammalian tissues. Fluorescent micrograph imagesof the mouse hippocampus show expression of miR-129-3p (Cy3) in tissuesections fixed with formaldehyde (a, Cy3), formaldehyde-BrCN solution in1-methylimidazole buffer at pH 8.0 (b, Cy3), formaldehyde-BrCN solutionin MES buffer at pH 7.0 (c, Cy3), formaldehyde-EDC solution diluted in1-methylimidazole buffer at pH 8.0 (c, Cy3) and formaldehyde-EDC-BrCN(d, Cy3). Scale bars 500 μm in a-e.

FIG. 11: Automation of formaldehyde-EDC miRNA ISH protocol. Fluorescentmicrograph images of the mouse cerebellum show expression of miR-124(Cy3) in tissue sections fixed with formaldehyde-EDC fixation (a, top,b, left Cy3), and miR-129-3p (c, left, Cy3). Nuclei were stained (DAPI,right, a-c). Scale bars 500 μm in b-c.

FIG. 12: miRNAs retained in formaldehyde+EDC fixed tissues.

FIG. 13: Comparison of formaldehyde/EDC and conventional formaldehydefixation for detection of a high and low abundance miRNA in mouse brainby ISH. (a-d) Fluorescence microscopy images of ISH using mouse braintissue sections fixed with formaldehyde/EDC (left) or conventionalformaldehyde alone (right), captured with identical camera settings andexposure times. Highly abundant nervous system specific miR-124 (clonecount frequency 8.8%) showed formaldehyde/EDC fixation moderatelyimproved signal intensity (a), compared to formaldehyde fixation alone(b). (c-d) Detection of lower expressed miR-130a (clone count frequency0.12%) using formaldehyde/EDC (c). Conventional formalin fixed samplesshowed no signal (d). The digoxigenin-labeled LNA-modified DNA probe washybridized to a specific miRNA and the signal was amplified using thetyramide-Cy3 detection system. Cell nuclei were counterstained with DAPI(blue; bottom panel). Scale bars were 200 μm.

FIG. 14: Images of miR-124 ISH show the majority of staining occurs inneurons. (a) Photomicrograph from ISH of miR-124 (Cy3) in mousehippocampus (see Supplemental methods for experimental details). (b)Prior to miRNA ISH, samples stained to visualize the localization ofneurons with a primary antibody for NeuN (Fluor488), a knownneuron-specific nuclear protein. (c) Nuclear staining (DAPI). (d)Overlay of miR-124 (red; Cy3), NeuN (Fluor488), and nuclear staining(DAPI). Scale bars, 200 μm (a-d). Note: double labeling for the miRNAISH and immunohistochemistry resulted in a weaker signal for both theimmunohistochemistry and miRNA ISH.

FIG. 15: Controls for miRNA probe specificity. (a) Images ofneuron-specific miR-124 ISH (Cy3) of fully complementary probes showrobust staining in the mouse cerebellum. miR-124 mismatch probesmiR-124-T19A (b) and miR-124-C3A (c) signal is reduced but visible.MiR-124 mismatched near the middle or seed region, miR-124-C9A, C10A andmiR-124-C9A respectively, show the least signal (d,e), similar tobackground (f). (g) Cistronically expressed miRNAs miR-99a (left, Cy3)and let-7c (center) exhibit similar band-like distributions in thecortex (Cy3; red), marked by arrows. Nuclei were counter-stained (right,DAPI; blue). (h) miR-140 (left, Cy3) and miR-140*(center, Cy3; red)co-localize in cortex and DAPI stain (left). Scale bars, 1.0 mm (a-f),500 μm (g), 200 μm (h). For images (a-e), ISH was performed at 47.5° C.,which was 20° C. below the T_(M) of the perfect probe/miR-124 duplexdetermined at 1.5 μM strand concentrations. For image (g), a second genecopy of let-7c (clone count frequency 4.1%) is present in the cluster ofmir-let-7c-2/mir-let-7b, which was approx. 2.5 fold more abundant thanmiR-let-7b (clone count frequency 1.7%).

FIG. 16: Fluorescence and formazan deposition NBT/BCIP detectionsystems. (a) Fluorescent image of mouse brain cerebellum usingTyramide-Cy3 detection system. (b) Light microscopy image of serialsections of mouse brain cerebellum using NBT/BCIP detection system showsimilar cellular distribution as in fluorescent detection (a). (c)Images of DAPI nuclear stain. Scale bars, 1.0 mm (a-c).

FIG. 17: Detection of miRNAs in mouse heart and liver sections usingformazan deposition NBT/BCIP detection system. (a) Images of a highlyabundant miR-122 ISH in the mouse liver sections, (b) the lowerexpressed miR-126, and a no probe control (c). (d) Images of the mouseheart sections show expression of highly abundant miR-1 (d), with a noprobe, negative control (e). Cell nuclei were counterstained with DAPI(blue; right panel (a-e); Scale bars, 200 μm (a-e).

FIG. 18: EDC derivatives. The water-soluble core of carbodiimides, whichis responsible for the crosslinking is shown bold.

DETAILED DESCRIPTION OF THE INVENTION Method for Fixing

In one aspect, the invention relates to a method for fixing a shortnucleic acid in a biological sample. The method includes contacting thebiological sample with an aldehyde-containing fixative, and subsequentlycontacting the sample with at least one of the following agents: awater-soluble carbodiimide or cyanogen bromide.

“Fixing” as used herein refers to immobilizing a short nucleic acidwithin the biological sample. “Immobilizing” as used herein refers tobinding the short nucleic acid to the biological sample such that thebinding is sufficient to be stable under conditions of washing, probing,labeling, and/or analysis.

A “nucleic acid” or “oligonucleotide” or “polynucleotide” refers to atleast two nucleotides covalently linked together. The nucleic acid maybe any type of nucleic acid, such as DNA or RNA. Exemplary nucleic acidsinclude mRNA, tRNA, rRNA, shRNA, siRNA or Piwi-interacting RNA, or apri-miRNA, pre-miRNA, miRNA, snoRNA, long ncRNAs, anti-miRNA, and anyvariants thereof. Additional nucleic acids include genomic DNA, cDNA, ora hybrid wherein the nucleic acid may contain combinations of deoxyribo-and ribo-nucleotides, and combinations of bases including uracil,adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine,isocytosine and isoguanine. Further examples of nucleic acids includeDNA or RNA of a virus, or nucleic sequences derived from a virus genome.In one embodiment, the nucleic acid is a short DNA or RNA moleculederived from a degraded source, such as, for example, degraded mRNA.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that may have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones. Nucleic acids containing one or more non-naturally occurringor modified nucleotides are also included within one definition ofnucleic acids. The modified nucleotide analog may be located for exampleat the 5′-end and/or the 3′-end of the nucleic acid molecule.Representative examples of nucleotide analogs may be selected fromsugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. TheT-OH-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH₂, NHR, NR₂ or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modified nucleotides and nucleic acids mayalso include locked nucleic acids (LNA), as described in U.S. PatentApplication No. 20020115080, which is incorporated herein by reference.

Modifications of the ribose-phosphate backbone may be done for a varietyof reasons, e.g., to increase the stability and half-life of suchmolecules in physiological environments, to enhance diffusion acrosscell membranes, or as probes on a biochip. Mixtures of naturallyoccurring nucleic acids and analogs may be made; alternatively, mixturesof different nucleic acid analogs, and mixtures of naturally occurringnucleic acids and analogs may be made.

A nucleic acid may include variants thereof. A “variant” as used hereinrefers to (i) a portion of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Nucleicacids may be synthesized as a single stranded molecule or expressed in acell (in vitro or in vivo) using a synthetic gene. Nucleic acids may beobtained by chemical synthesis methods or by recombinant methods.

A nucleic acid also encompasses the complementary strand of a depictedsingle strand. Many variants of a nucleic acid may be used for the samepurpose as a given nucleic acid. Thus, a nucleic acid also encompassessubstantially identical nucleic acids and complements thereof. A singlestrand provides a probe that may hybridize to a target sequence understringent hybridization conditions. Thus, a nucleic acid alsoencompasses a probe that hybridizes under stringent hybridizationconditions.

A “short” nucleic acid refers to a nucleic acid that has a maximumnumber of base pairs in length of about 100, 90, 80, 70, 60, 50, 40, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 bp. The shortnucleic acid has a minimum number of base pairs in length of about 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bp. Any minimumamount can be combined with any maximum amount to define a range for ashort nucleic acid.

Exemplary short nucleic acids include mRNA, tRNA, shRNA, siRNA orPiwi-interacting RNA, or a pri-miRNA, pre-miRNA, miRNA, and anti-miRNA,or any variants thereof. In one embodiment, the target polynucleotide isa short DNA or RNA molecule derived from a degraded source, such as, forexample, degraded mRNA.

In a preferred embodiment, the short nucleic acid is microRNA (miRNA).MicroRNA molecules are known in the art (see, for example, Bartel, Cell,2004, 116, 281-297 for a review on microRNA molecules). The definitionsand characterizations of microRNA molecules in the article by Bartel arehereby incorporated by reference. Such molecules are derived fromgenomic loci and are produced from specific microRNA genes.

miRNAs are typically small RNA molecules of generally about 13-33,18-24, or 21-23 nucleotides in length. The miRNA may also have a totalof at about 5-40 nucleotides in length. These microRNAs are non-codingRNAs which are cleaved from hairpin precursors. miRNAs are naturally 5′phosphorylated and carry 2′, 3′ dihydroxyl termini. The sequence of themiRNA may comprise the sequence of a miRNA disclosed in U.S. patentapplication Ser. Nos. 11/384,049, 11/418,870 or 11/429,720, the contentsof which are incorporated herein, or variants thereof.

A source of the short target nucleic acid is a biological sample. A“biological sample” as used herein refers to a sample of biologicaltissue or fluid that includes biomolecules. Such samples include, butare not limited to, tissue or fluid isolated from animals or plants.Biological samples also include viruses or unicellular organisms.Biological samples may also include sections of tissues such as biopsyand autopsy samples, frozen sections taken for histologic purposes,hair, and skin. Biological samples also include explants and primaryand/or transformed cell cultures derived from animal or patient tissues.Biological samples may also be blood, a blood fraction, plasma, serum,urine, pleural effusion, mucus, ascitic fluid, amniotic fluid, stool,tears, saliva, cerebrospinal fluid, cervical secretions, vaginalsecretions, endometrial secretions, gastrointestinal secretions,bronchial secretions, sputum, secretions from ovarian cyst, sperm,secretions from the breast, cell line, or tissue sample.

A biological sample may be provided by removing a sample of cells froman animal, or plant, but can also be accomplished by using previouslyisolated cells (e.g., isolated by another person, at another time,and/or for another purpose), or by performing the methods describedherein in vivo. “Animal” as used herein refers to any animal, includingfish, amphibians, reptiles, birds, and mammals, such as mice, rats,rabbits, goats, cats, dogs, cows, apes and humans.

As used herein, the term “biomolecule” in the context of a molecule thatbinds to a short nucleic acid refers to an entity that can or does bindto a short nucleic acid. Biomolecules include biological molecules, suchas a protein, nucleic acid, carbohydrate, fat, and lipid. Exemplaryshort nucleic acid-binding biomolecules include polypeptides, nucleicacids, small molecules such as hormones, cytokines, and drugs. In onepreferred embodiment, the biomolecule is a nucleic acid.

Contacting the Biological Sample with a Fixative

The method includes contacting the biological sample with analdehyde-containing fixative under conditions in which a biomoleculecovalently bonds to a nucleic acid, as is known in the art. Such methodsare known in the art. See, e.g., Feldman, “Reactions of nucleic acidsand nucleoproteins with formaldehyde,” Prog. Nucleic Acid Res Mol. Biol.1973, 13:1-49, which is incorporated by reference.

The aldehyde-containing fixative can include an aldehyde-based fixativealone, or in combination with other fixative agents. Exemplary fixativeagents include osmium tetroxide, picric acid, dialdehyde starch, AEDP(3-[(2-Aminoethyl)dithio]propionic acid.HCl) ethanol, Ketones,Isocyanate-containing compounds to label hydroxyl-containing molecules,Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3′-sulphonate),1,1′-Carbonyldiimidazole (CDI),Bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl]sulfone (BSOCOES) andsulfo-BSOCOES, Ethylene glycol-bis(succinic acid N-hydroxysuccinimideester) EGS, Sulfo-EGS, N,N′-Disuccinimido Carbonate (DSC), Imidoester.Modification of the 5′ phosphate using EDC and other coupling reagents,Cystamine followed by DTT and a sulfhydryl crosslinker. (N-Succinimidyl3-(2-pyridyldithio)-propionate) and LC-SPDP (Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP modification ofamines coupled to the 5′ phosphate of nucleotides followed by a reducingagent, DTT, creating a sulfhydryl group attachment, and a sulfhydrylcrosslinker. SATA to modify a 5′-amine derivative of oligonucleotides,forming a protected sulfhydryl for crosslinking.

Examples of an aldehyde-based fixative agent includes, for example,formaldehyde, a derivative of formaldehyde, paraformaldehyde, glyoxal,and glutaraldehyde.

Contacting the Biological Sample with a Water-Soluble Carbodiimide orCyanogen Bromide

Subsequent to the step of contacting a biological sample with analdehyde-containing fixative, the method includes contacting the samplewith at least one of the following agents: a water-soluble carbodiimideor a cyanogen halide. A water-soluble carbodiimide is preferred.

Exemplary water-soluble carbodiimides include1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC),1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide Hydrochloride,1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), andN,N′-diisopropylcarbodiimide (DIC), and derivatives thereof. Examples ofderivatives of water-soluble carbodiimides include those illustrated inFIG. 18. In one embodiment, the water-soluble carbodiimide is1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC).

Examples of cyanogen halides include cyanogen bromide, cyanogenfluoride, cyanogen chloride, cyanogen iodide. Cyanogen bromide ispreferred.

In another embodiment, the method includes contacting the sample with awater-soluble carbodiimide, followed by a cyanogen halide. For example,the method may include contacting the sample with an aldehyde-containingfixative, then subsequently contacting the sample with1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide or1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide Hydrochloride, then withcyanogen bromide.

In a further embodiment, the water-soluble carbodiimide and/or cyanogenhalide are in solution with a buffer. An exemplary buffer for awater-soluble carbodiimide includes 1-methylimidazole buffer, and anexemplary buffer for a cyanogen halide includesN-morpholinoehanesulfonate (MES) buffer.

The water-soluble carbodiimide and/or cyanogen halide solution has a pHgreater than 6.0 and less than 9.0. Preferably, the pH of thewater-soluble carbodiimide and/or cyanogen halide solution has a pH ofabout 7.0 to 8.5. In a preferred embodiment, the water-solublecarbodiimide is EDC and the pH of the EDC solution is about 8.0. Inanother preferred embodiment, the cyanogen halide solution has a pH ofabout 7.0.

In one embodiment, the water-soluble carbodiimide has a concentration ofabout 50 mM to about 250 mM. In another embodiment, the water-solublecarbodiimide contacts the sample at a temperature of about 20° C. toabout 70° C.

In one embodiment, the cyanogen halide has a concentration of about 10mM to about 500 mM. In another embodiment, the cyanogen halide contactsthe sample at a temperature of about 0° C. to about 40° C.

Method for Detecting

In another aspect, the invention relates to a method for detecting ashort target nucleic acid in a biological sample. The method includescontacting the biological sample with an aldehyde-containing fixativeand subsequently contacting the sample with a water-soluble carbodiimideor cyanogen bromide to produce a cross-linked short nucleic acid. Themethod further includes contacting the cross-linked short nucleic acidwith a probe, said probe being complementary to all or a part of aregion of interest of the short nucleic acid, thereby producing ahybridized short nucleic acid. The method also includes detecting thehybridized short nucleic acid as the short target nucleic acid.

“Detecting” refers to determining the presence of a component in asample. Detection may also mean determining the absence of a component.Detection may also mean measuring the level of a component, eitherquantitatively or qualitatively.

A “target nucleic acid” as used herein refers to any nucleic acid thatis to be identified, fixed, or otherwise analyzed. The target nucleicacid may be any type of nucleic acid, such as DNA or RNA. Exemplarytarget nucleic acids include DNA or RNA of a virus, or nucleic sequencesderived from a virus genome. Exemplary target nucleic acids includemRNA, tRNA, shRNA, siRNA or Piwi-interacting RNA, or a pri-miRNA,pre-miRNA, miRNA, and anti-miRNA, or any variants thereof. In oneembodiment, the target polynucleotide is a short DNA or RNA moleculederived from a degraded source, such as, for example, degraded mRNA.

As stated above, subsequent to fixing or cross-linking, the methodincludes contacting the cross-linked short nucleic acid with a probe. Inone embodiment, one or more than one probe may be used to bind to thetarget nucleic acid.

In a preferred embodiment, the probe is a polynucleotide of single- ordouble-stranded DNA or RNA. For example, a probe may be single strandedor partially single and partially double stranded. The strandedness ofthe probe is dictated by the structure, composition, and properties ofthe target sequence.

The probe is capable of binding to a target nucleic acid ofcomplementary sequence or a substantially complementary sequence throughone or more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. Probes may bind totarget nucleic acid without complete complementarity to the probesequence, depending upon the stringency of the hybridization conditions.

In a preferred embodiment, the probe has 100% complementarity to all ora portion of a region of interest of the target nucleic acid.“Complement” or “complementary” as used herein refers to Watson-Crick(e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides ornucleotide analogs of nucleic acid molecules.

“Substantially complementary” used herein may mean that a first sequenceis at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the complement of a second sequence over a region of2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-40,40-60, 60-100, or more nucleotides, or that the two sequences hybridizeunder stringent hybridization conditions.

The site on the target nucleic acid on which the probe binds is the“target binding site.” The target binding site may be 5-100 or 10-60nucleotides in length. The target binding site may include a total of 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30-40, 40-50, 50-60, 61, 62 or 63 nucleotides. Anyminimum amount can be combined with any maximum amount to define a rangefor a target binding site.

The probe is preferably contacted to the biological sample understringent hybridization conditions. “Stringent hybridization conditions”used herein may mean conditions under which a first nucleic acidsequence (e.g., probe) will hybridize to a second nucleic acid sequence(e.g., target nucleic acid), such as in a complex mixture of nucleicacids. Stringent conditions are sequence-dependent and will be differentin different circumstances. Stringent conditions may be selected to beabout 5-10° C. lower than the thermal melting point (T_(M)) for thespecific sequence at a defined ionic strength pH. The T_(M) may be thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(M), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.5and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

The DNA or RNA probe may have a length of from 8 to 500, 10 to 100 or 20to 60 nucleotides. The probe may also have a length of at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280 or 300 nucleotides. The probe is preferablyabout 10 to 30 nucleotides in length, more preferably 18-25 nucleotidesin length. Any minimum amount can be combined with any maximum amount todefine a range for a probe.

The probe may be directly labeled or indirectly labeled. A “label” asused herein refers to a composition that is detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, and/or physicalmeans. For example, suitable labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and other molecules that can be madedetectable.

The label may be a fluorophore, such as described in U.S. Pat. No.6,541,618. The label may also be a quencher molecule, which when inproximity to another label, may decrease the amount of detectable signalof the other label, such as described in U.S. Pat. No. 6,541,618. Thelabel may be incorporated into nucleic acids and proteins at anyposition of the nucleic acids or proteins.

Examples of direct labeling, such as Chemical labeling, include KreatechULS chemical labeling technology, which labels miRNA or probes or targetnucleic acids with biotin. Another example of direct labeling is part ofa PerkinElmer Micromax™ Direct Labeling Kit, which labels miRNAs withbiotins along the length of the miRNA. An example of enzymatic Endlabeling includes ligation of dinucleotides with a biotin entity(pCU-bio). Signal amplification, such as Tyramide signal amplification(TSA) amplifies the number of biotins in site, starting from one biotinto which a Streptavidin-horse radish peroxidase (HRP) conjugate isbound. The Tyramide biotin substrate is processed by the HRP to producea non soluble biotin that is precipitated in site, creating a cluster ofbiotins on the appropriate microsphere.

In one embodiment, the probe includes a locked nucleic acid (LNA), asdescribed in U.S. Patent Application No. 20020115080, which isincorporated herein by reference. Exemplary probes include about 5 to 8LNAs.

The probe may further include a linker. The linker may be 10-60nucleotides in length. The linker may be 20-27 nucleotides in length.The linker may be of sufficient length to allow the probe to be a totallength of 45-60 nucleotides. The linker may not be capable of forming astable secondary structure, may not be capable of folding on itself, ormay not be capable of folding on a non-linker portion of a nucleic acidcontained in the probe. The sequence of the linker may not appear in thegenome of the animal from which the probe non-linker nucleic acid isderived.

In addition to contacting the cross-linked short nucleic acid with aprobe, the method may further include contacting the biological samplewith a probe complementary to all or a part of a region of interest ofanother target nucleic acid in the biological sample, thereby producinga hybridized target nucleic acid. Accordingly, in one embodiment, morethan one target nucleic acid may be hybridized with a probe andidentified. In a further embodiment, multiple probes with differentlabels can be hybridized to different target nucleic acids. For example,a probe can be used that hybridizes to a miRNA of interest, concurrentlywith another probe that hybridizes to a short nucleic acid that isdegraded mRNA and/or an mRNA variant. The identification of a specificprobe or a combination of different probes can be used to identify thephenotype of the cell, for example whether the biological sample is atype of cancer.

Kit

In another aspect, the invention relates to a kit for fixing a shortnucleic acid in a biological sample. The kit includes a supportsubstrate for holding the sample, an aldehyde-containing fixative, andat least one the following agents: a water-soluble carbodiimide orcyanogen bromide.

A “support substrate” refers to a composition that is amenable to atleast one detection method and contains individual sites that areappropriate for attachment or association of the biological sample,probe, and nucleic acid. Exemplary support substrates include glass,modified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon J,etc.), polysaccharides, nylon or nitrocellulose, resins, silica orsilica-based materials including silicon and modified silicon, carbon,metals, inorganic glasses and plastics. The substrates may allow opticaldetection without appreciably fluorescing.

Preferably, the support substrate is one to which the biological samplemay be bound. The binding of the biological sample to the supportsubstrate may be covalent or non-covalent. Covalent bonds may be formeddirectly between the probe or biological sample and the solid support ormay be formed by a cross-linker or by inclusion of a specific reactivegroup on either the solid support or the probe or both molecules.Non-covalent binding may be one or more of electrostatic, hydrophilic,and hydrophobic interactions. Included in non-covalent binding is thecovalent attachment of a molecule, such as streptavidin, to the supportand the non-covalent binding of a biotinylated probe to thestreptavidin. Immobilization may also involve a combination of covalentand non-covalent interactions.

The kit may also include any or all of the following: assay reagents,buffers, probes and/or primers, and sterile saline or anotherpharmaceutically acceptable emulsion and suspension base. In addition,the kits may include instructional materials containing directions(e.g., protocols) for the practice of the methods described herein. Forexample, the kit may be a kit for the fixation, amplification,detection, identification or quantification of a target nucleic acidsequence.

Other aspects of the invention will become apparent to the skilledartisan by the following description of the invention.

EXAMPLES

The following examples are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

General Methods Microscopy and Image Processing

Images were captured on an Olympus BX50 microscope equipped with a DP70camera and Olympus DP controller software. For florescent imaging weused the following filter sets; U-MWU2 (Olympus) for DAPI, 41001 HQ(Chroma) for Fluro488, 49004 ET (Chroma) for Cy3. The images from boththe immunohistochemistry and the ISH were captured using DP70 camera andprocessed using Olympus Microsuite Five software (Olympus).

Northern Blotting

Northern blotting was performed as described1 using Hybond-N+membrane(Amersham GE healthcare), and the hybridization and wash steps wereperformed at 50° C. The oligodeoxynucleotide probes were 5′-labelledwith [γ-32] ATP. The probe for miR-124 was 5′ TTGGCATTCACCGCGTGCCTTA. Tocontrol for loading of the gel, 5S rRNA was detected by ethidium bromidestaining of the polyacrylamide gel prior to transfer. Probed sampleswere recorded by phosphoimaging and quantified. Northern blotting imageswere quantified using ImageJ software5.

miRNA Cloning and Sequencing

RNA was extracted from dissected mouse brain regions andsize-fractionated by a denaturing PAGE. miRNA cloning was performed asdescribed previously6. The cDNA library was sequenced by 454 sequencing.miRNA sequences were annotated as described1 and obtained about 90,000sequence clones.

Supplemental Methods Design and Synthesis of LNA-ModifiedOligodeoxynucleotide Probes

LNA-spiked oligodeoxynucleotide probes (LNAs) were identical in lengthand fully complementary to the predominantly cloned mature miRNA ormiRNA* sequence1. On average, we placed 5 to 8 LNA residues in a 22-ntoligodeoxynucleotide probe. To optimize probe hybridization, we avoidedinserting LNA bases in positions that may stabilize internal secondarystructures, so LNA residues were placed outside of predicted secondaryself-structures. The LNA probe sequences are listed in SupplementaryTable 1. Probes were synthesized at 0.2 or 0.4 μmol scale on an ABI 3400DNA synthesizer using 3′-amino-modifier C7 CPG (500 Å) solid glasssupport (Glen Research) and LNA and DNA phosphoramidites (Sigma-Proligoand Glen Research). The aminolinker Fmoc and phosphate cyanoethylprotecting groups were removed by gently passing back and forth 3 ml offreshly prepared 20% piperidine (Sigma) in N,N-dimethylformamide (DMF,Sigma) for 5 min over each column. Columns were washed three times with3 ml of acetonitrile and dried with pressurized air. For furtherdeprotection, the CPG was transferred to a 1.5 ml screw cap tube andincubated with 1.2 ml of 28% aqueous ammonium hydroxide solution for 16h at 55° C. The tube was placed on ice for 5 min, and the supernatanttransferred to a 13 ml centrifugation tube. 10 ml of 1-butanol wasadded, vigorously mixed and the LNA pellet was collected bycentrifugation at 13,000 rpm in an SS-34 rotor at 4° C. for 20 min inSorvall RC5C Plus centrifuge. The supernatant was removed completely,the pellet dried in an Eppendorf Vacufuge concentrator and redissolvedin 282 μl water. In order to exchange ammonium ions with sodium, the LNAsolution was adjusted to 0.3 M NaCl by addition of 18 μl of 5 M NaCl andprecipitated by addition of 900 μl of 100% ethanol. The pellet wascollected by centrifugation and redissolved in 300 μl water. The LNAconcentration was determined by measuring its UV absorbance at 260 nmusing an average absorbance coefficient of 11,000 M-1 cm⁻¹ pernucleotide.

To 3′-digoxigenin-label the LNA probe, an aliquot corresponding to 30nmol of the LNA was dried in an Eppendorf concentrator and redissolvedin 50 μl of 100 mM sodium carbonate buffer at pH 8.5. 240 nmol ofdigoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimideester (Roche) freshly dissolved in 50 μl anhydrous DMF was added, mixedand incubated for 16 h at 25° C. The yield of a typical labelingreaction was 80%. The DIGlabeled probes, which showed reduced mobilitycompared to the starting material, were separated on a 43×23×0.2 cmdenaturing 18% PAGE gel (200 ml gel volume) for 6 h at 50 W. Productbands were visualized by UV shadowing, excised, and eluted from the gelslice overnight into 3 volumes (w/v) of 0.3 M NaCl, ethanol precipitatedand resuspended in 100 μl water.

DIG-labeling was examined by spotting 1 μmol, 100 fmol and 10 fmol on aNylon membrane (Amersham Hybond—N+) followed by incubation of themembrane with anti-DIG antibody fragment conjugated to peroxidaseantibody (Roche) with NBT/BCIP (Roche). Formazan Blue deposition isobserved between 10 min to 1 h for all amounts spotted.

UV Melting Profiles of miRNA-LNA-Probe Duplexes

The UV absorbance change associated with melting of the LNA probe andmiRNA duplex was recorded at 280 nm on an Uvikon UV/VISspectrophotometer (LifePower software) equipped withtemperature-controlled cuvette holders. The rate of cuvette heating andcooling was 0.3° C./min and the absorbance was recorded every min. Themelting temperature (TM) of duplex formation was obtained bycurve-fitting the absorbance change as a function of temperature to atwo-state model using Meltwin 3.5 software. To prepare samples for T_(M)analysis, miRNA+probe pairs in a volume of 300 μl of 1.5 μM of unlabeledLNA probe and 1.5 μM of synthetic miRNA in 750 mM NaCl, 75 mM sodiumcitrate, 50 mM sodium phosphate (pH 7.0) and 50% formamide wereincubated for 5 min at 95° C., for 5 min at 80° C., gradually cooled for3 h to 50° C., and then held at room temperature for 1 h or longer. Thesolution was then transferred to a quartz cuvette, overlayed with 500 μlmineral oil (Sigma) and degassed for 10 min by applying a vacuum to asmall dessicator holding the cuvettes.

Adjusted Parameters for Prediction Programs to Estimate T_(M) formiRNA-LNA Probe Duplex in Formamide Containing ISH Buffer.

To best emulate our experimental T_(M) analysis and introduce acorrection factor, we tested several salt concentrations in theprediction program (lna-tm.com) and determined that 50 mM salt, insteadof the actual 750 mM present in ISH buffer, yielded very similar valueswith an average predicted T_(M) of 0.26° C. higher than the average ofour experimental values with a standard deviation of 4.4° C., based on asample of 127 miRNA-LNA probe duplex melting profiles. To deriveestimates for the likelihood that the predicted T_(M) error will fallwithin a specified range, we constructed nonparametric predictionintervals. We found that with 95.3% probability, the error in thepredicted T_(M) for any miRNA-LNA probe pair will fall between −9.2° C.and 8.6° C. With 68.8% probability, the predicted T_(M) error will fallbetween −4.1° C. and 4.7° C. Within a broad range of ±2 standarddeviations of the mean, the sampled distribution of predicted T_(M)errors is closely approximated by a normal distribution with μ=0.26° C.and σ=4.4° C. We therefore repeated the same analysis under theassumption of normality and found closely similar results. We found thatwith a 95.3% probability, the error in the predicted T_(M) for anymiRNA-LNA pair falls within −9.2° C. and 8.6° C. of the experimentalT_(M) in formamide-containing ISH buffer or a 68% probability that thetrue T_(M) will fall between −3° C. and 5.1° C.

Tissue Preparation and Processing

Male, 2-month-old C57BL/6J mice (Jackson Labs) were maintained on a 12 hlight/dark cycle. Animals were sedated in accordance with NIH AnimalWelfare guidelines using ketamine and xylazine cocktail before organperfusion with 50 ml of Tris-HCl buffered Saline (TBS) containing 50 mMTris-HCl, 150 mM NaCl and the pH adjusted to 7.4. Immediately after TBS,we perfused with 30 ml of 4% paraformaldehyde (PFA) in TBS. Tissues werecollected, immersed in 30 ml of 4% PFA in TBS for 24 h at 4° C., then in30 ml of 0.5 M sucrose diluted in TBS at 4° C. for 48 h. The tissueswere mounted in Tissue-Tek OCT Compound, frozen in a dry-ice+ethanolbath in a Cryomold (Tissue-Tek), immediately serial sectioned from 5 to40 μm with a cryostat (Leica) and mounted on SuperFrost Plus glassslides (Thermo Fisher Scientific). Unprocessed specimen or mountedslides can also be stored at −80° C.

In Situ Hybridization Procedure

Processing or wash steps were generally carried out by placing up to 20slides in 75 ml glass Coplin jars (Electron Microcopy Sciences) filledwith specified solutions and all wash steps were in 50 ml of the statedsolution for 5 min at 25° C., unless otherwise noted. Tissue sectionsmounted on glass slides were thawed and air-dried for 1 h at 25° C. andthen incubated in a 75 ml solution containing 20 μg/ml proteinase K(Roche) in TBS, pH 7.4 for 20 min. Thereafter, the slide was washed twotimes in 75 ml TBS. Samples were fixed in 75 ml of 4% PFA in TBS for 10min, washed with 0.2% (w/v) glycine in TBS, and washed two times in 75ml TBS. To remove residual phosphate from the TBS washes, slidesprocessed with EDC fixation were incubated twice for 10 min in 75 ml ofa freshly prepared solution containing 0.13 M 1-methylimidazole, 300 mMNaCl, pH 8.0 adjusted with HCl. To prepare 160 ml of imidazole buffer,add 1.6 ml of 1-methylimidazole to 130 ml water, adjust the pH by addingapproximately 450 μl 12 M HCl to pH 8.0, then add 16 ml 3 M NaCl andwater to a final volume of 160 ml. In the meantime, prepare a solutionof 0.16 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Sigma) byadding 176 μl EDC into 10 ml of 1-methylimidazole+300 mM NaCl (pH 8.0)solution, and then readjust the pH of the EDC solution by furtheraddition of approximately 100 μl 12 M HCl to pH 8.0. It is important forstorage of commercial EDC to handle the reagent under anhydrousconditions and protect it with a layer of argon. Aqueous solutionscontaining EDC and/or 1-methylimidazole cannot be stored. Place slidesin a humidified chamber and add 500 μl of EDC solution to each slide andincubated for 1 to 2 h at 25° C. The slides were washed in 0.2% (w/v)glycine/TBS and then washed twice in TBS.

For inactivation of enzymes in tissues, e.g. alkaline phosphates andperoxidases, slides were acetylated by incubating for 30 min in a 75 mlsolution of freshly prepared 0.1 M triethanolamine and 0.5% (v/v) aceticanhydride. Slides were then rinsed twice in TBS. For pre-hybridization,the tissue sections on the slides were covered with 500 μl ofhybridization buffer containing 50% formamide, 5×SSC, 5×Denhardt'ssolution (Applichem), 250 μg/ml yeast tRNA (Sigma), 500 μg/ml salmonsperm DNA (Sigma), 2% (w/v) Blocking Reagent (Roche), 0.1%3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPs)(Sigma), 0.5% Tween at 25° C. for 2 h in a humidified chamber. Thehybridization buffer was removed by tilting the slide.

For hybridization, 4 μmol of DIG-labeled LNA probe diluted in 100 μl ofhybridization buffer were applied per section, and covered withcoverslips (LifterSlips, Fisher). The slides were incubated in a sealedhumidified chamber for 16 h at a temperature 20° C. below the T_(M) ofthe experimentally determined miRNA-LNA probe duplex. Hybridizationconditions were adjusted 20° C. below the LNA probe's experimentallydetermined T_(M), taking into account that the probe concentration foran ISH experiment is 40 nM compared to 1.5 μM required for optical

T_(M) Measurements.

The slides were immersed in 20 ml of 5×SSC at 25° C. so the coverslipcan be removed and then washed twice in 75 ml of a solution containing50% formamide, 1×SSC, and 0.1% Tween for 30 min at the same temperatureas probe hybridization. Finally slides were washed in 75 ml 0.2×SSC for15 min and once in 75 ml of 0.1% Tween in TBS. To inactivate endogenousperoxidase activity, slides were incubated in 75 ml of 3% hydrogenperoxide in TBS with 0.1% Tween for 30 min, followed by three 1 minwashes of TBS/0.1% Tween.

In preparation for probe detection, 500 μl of blocking solutioncontaining 0.5% Blocking Reagent (Roche), 10% heat inactivated goatserum, and 0.1% Tween 20 in TBS was applied to each slide for 1 h at 25°C., then incubated in anti-DIG-FAB peroxidase (POD) (Roche) diluted1:500 in blocking solution for 1 h at 25° C. Slides were then washed asdescribed below in preparation for the application of the variousdetection reagents.

Cy3 Fluorescent Detection System

The slides were washed twice in a solution containing 0.1% Tween 20, inTBS and 200 μl of TSA Plus Cy3 System working solution was applied ontoto the sections for 10 min at 25° C. in the dark according to themanufacturer's protocol (PerkinElmer Life Sciences). The slides werethen washed three times in TBS with a tilting rotator. The slides weremounted using 2 drops of Vectashield mounting medium with DAPI (VectorLaboratories) and samples processed for microscopy.

Alkaline Phosphatase Double Amplification Detection System

This detection method was adapted from studies by Lein et al. The slideswere washed three times in 75 ml in TNT buffer, consisting of 0.1 MTris-HCl, pH 7.5, 0.15 M NaCl, and 0.1% Tween 20. Thereafter, 250 μl ofbiotinylated tyramide solution from the Individual Indirect TyramideReagent kit (PerkinElmer Life Sciences) was applied to each slide for 30min at 25° C., according to the manufacturer's protocol. The slides werewashed three times in 75 ml maleate buffer (0.09 M maleic acid, 0.175 MNaOH, 1 M NaCl, 0.5% Tween 20, pH 7.5). A 1:500 solution of 350 μl ofNeutrAvidin-conjugated alkaline phosphatase (Thermo Scientific) inmaleate buffer supplemented with 10 mg/ml blocking reagent (Roche) wasapplied to the slide for 40 min at 25° C. Slides were then washed twicein 75 ml maleate buffer, and four times in 75 ml TMN buffer (0.1 M Trisbase, pH 9.5, 0.05 M MgCl2, 0.5 M NaCl, 0.5% Tween 20, 2 mM(−)-tetramisole hydrochloride). The formazan deposition was performed byapplying 200 μl of a solution containing 0.375 mg/ml Nitro bluetetrazolium chloride (NBT) (Roche) and 0.188 mg/ml of5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt (BCIP) (Roche) inTMN buffer. Upon deposition of the blue pigment, typically visible after30 min, the slides were washed twice in 75 ml water, three times in 75ml of 0.01 M Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM EDTA, 0.05% Tween 20,and incubated for 10 min in 75 ml of 4% aqueous paraformaldehydesolution. Finally, the slides were washed in 75 ml water and mountedusing 2 drops of Vectashield mounting medium with DAPI (VectorLaboratories).

Immunohistochemistry and miRNA ISH Co-Staining

To assess whether miR-124 was localized specifically to neurons, brainsections were processed for double immunofluorescence staining tovisualize the simultaneous localization of miR-124 (red; Cy3) and aprimary antibody for NeuN (green; TRITC), a known neuron-specificnuclear protein. First we performed immunohistochemistry to labelneuronal cells. Brain sections mounted on slides were washed three timesfor 5 min in 50 ml of TBS. Sections were incubated for 1 h in 500 μl of“working solution of M.O.M. Mouse Ig Blocking Reagent” (VectorLaboratories) at 25° C., according to the manufacturer's protocol.Sections were washed twice for 5 min in 50 ml of TBS, followed byincubation in a “working solution of (M.O.M.) diluent” (VectorLaboratories) for 5 min at room temperature, then the “working solutionof (M.O.M.) diluent” was removed. To visualize neurons, we incubated thesamples with a primary anti-NeuN-clone-A60-AlexaFluor488 conjugatedantibody (Chemicon International, Temecula, Calif.) diluted 1:300 in a“working solution of M.O.M. diluent” (Vector Laboratories) for 1 h at25° C. Slides were then washed 3 times for 5 min in 50 ml of TBS. Slideswere mounted using 2 drops of Vectashield mounting medium with DAPI(Vector Laboratories). Please note that the glass coverslips were notsealed, as the samples were later used for ISH to detect miR-124. Afterimage acquisition, coverslips were removed by washes in 50 ml of TBS andthe tissue sections were then processed for ISH for miR-124 (Cy3; red)by applying our miRNA ISH protocol described earlier. After miRNA ISH,the sections were mounted using 2 drops of Vectashield mounting mediumwith DAPI (Vector Laboratories) and samples processed for microscopy.

Photomicrographs of the nuclear staining are required to superimposeimages for the immunohistochemistry and the miR-124 ISH. Images werecombined to generate an overlay using Olympus Microsuite Five software.Please note that the immunohistochemistry procedure for NeuN proteindetection somewhat reduced the efficiency for miRNA detection;performing miRNA ISH before protein staining was not possible as EDCfixation interfered with protein immunohistochemistry.

Small RNA Isolation from Formalin-Fixed Tissue

Formalin-fixed brain tissues used for RNA extraction were cut into 1 mmsections and incubated with a 200 μl solution containing 20 μg/mlproteinase K (Roche) in TBS and incubated at 45° C. for 1 h. The sampleswere then processed for RNA isolation. Total RNA was isolated from 4%formaldehyde fixed tissue by using the commercial Trizol (Invitrogen)reagent. We added 1 ml of Trizol per 1 mm slice of brain. The mixturewas immediately homogenized while kept on ice. Cells were homogenizedusing a Dounce glass homogenizer (Kimble-Kontes) and then furthermechanically homogenized using a Polytron homogenizer (Kinematica AG).Following tissue disruption, 1/10 volume of 3 M sodium acetate (pH 4.2)was added. The mixture was transferred to an Eppendorf tube andcentrifuged for 15 min at 12,000 g at 4° C. The aqueous phase wastransferred to a new tube without the white interphase. RNA was furtherextracted with 1/2 volume of acid-buffered phenol:chloroform:isoamylalcohol (25:24:1) and centrifuged for 15 min at 4° C. at 12,000 g.Finally, we extracted the aqueous phase with 1/2 volume chloroform. Theupper (aqueous) phase was transferred to a new tube. We added 3 volumesof ethanol to precipitate the RNA and incubated the sample overnight at−20° C. The RNA pellet was collected by centrifugation for 15 min at 4°C. at 12,000 g and the supernatant removed. The pellet was thendissolved in 30 μl water and used for Northern Blotting.

We were unable to isolate miR-124 from the formalin+EDC-treated tissue,presumably because miRNAs remained crosslinked to the protein matrix andthe small RNAs were lost to the phenol- or inter-phase during RNAisolation.

Example 1

To better understand the technical challenges associated with miRNA ISH,we investigated the importance of miRNA fixation and probehybridization. For fixation of proteins and nucleic acids in tissues, asolution containing 3.7% formaldehyde (10% formalin) is commonly used.Formaldehyde crosslinks are reverted by incubation at elevatedtemperature and this process is facilitated by proteinase K treatment.Reversal of the formaldehyde-based nucleic acid base modifications isalso necessary for probe hybridization, but it creates the problem ofmiRNA release and diffusion out of the tissue sections. Therefore, weexamined the extent of miRNA escape from tissue during ISH. We conducteda mock ISH for conventionally fixed brain, isolated RNA from the tissuesections and the ISH buffer, and probed both fractions for the highlyexpressed neuronal miR-124 by Northern blotting. At hybridizationtemperatures above 40° C., at least 50% of miR-124 initially present inthe tissue section accumulated in the buffer as early as 1 h (FIG. 1a-c). At 4° C., the hybridization buffer showed no signal for miR-124(FIG. 1 d), which would suggest that RNA-protein crosslinks remainedintact at lower temperatures.

To prevent the loss of miRNAs, we added an additional miRNA fixationstep that uses the miRNA 5′ phosphate end, which does not react withformaldehyde. The water-soluble1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC, reacts withphosphate and condenses it with amino groups in the protein matrix toform stable linkages. When formaldehyde-fixed specimens wereadditionally treated with EDC, miR-124 no longer escaped from sections,and only trace amounts of miR-124 were detected in the ISH buffer (FIG.1 e). EDC treatment alone, without prior formaldehyde fixation failed toretain miRNAs in tissues (data not shown).

Example 2

Probes that bind to miRNAs with high thermodynamic stability such as DNAprobes containing locked nucleic acid (LNA) residues are well suited formiRNA ISH. The melting of LNA-miRNA duplexes can be observedexperimentally by UV spectrophotometry. Thermodynamic analysis ofLNA-modified deoxynucleotide duplexes led to models that predict theirmelting temperatures (T_(M)) for any LNA-RNA pair. Unfortunately theT_(M)s cannot be predicted accurately for ISH, as the programs do notconsider the presence of formamide denaturant. To derive optimalhybridization conditions, nucleic acid melting studies were conductedand the melting profiles of 130 miRNA-LNA probe pairs were measured.With these data, we provide a correction factor for these programs, bymanipulating the salt parameter of LNA-RNA T_(M) prediction programs,and obtained useful starting points for estimating hybridizationtemperature in formamide-containing ISH buffer. We generated our LNAprobe set based on miRNAs expressed with a clone frequency of at least0.04% based on small RNA library sequencing from 5 regions of the mousebrain.

To determine the range of the ISH sensitivity, we conducted ISH formiRNAs identified at varying frequencies from small RNA cloninglibraries in mouse brain; miR-9, 9.3%; miR-124, 8.8%; miR-26a, 3.3%;miR-26b, 0.25%; miR-370, 0.14%; miR-130a, 0.12%; and miR-410, 0.07%.Tissue sections were fixed with formaldehyde+EDC and then ISH performed.We determined the effect of formaldehyde+EDC versus formaldehydefixation alone by conducting ISH for the most abundant miR-124 and the73-fold less abundant miR-130a. ISH images show that signal for miR-124were improved moderately, but miR-130a signals were only detectable informaldehyde+EDC-fixed samples (FIG. 13). Since the abundant miR-124presumably retained enough miRNA to nearly saturate the signal, theimprovements remained modest, but for lowly expressed miRNAs, loss bydiffusion hindered its detection in formaldehyde alone samples. miR-124is mostly detected in neuronal cells present in different regions of thebrain (FIG. 2 a-d and FIG. 14) and predominately localized in thecytoplasm, while the cell nuclei were excluded. Another highly expressedmiRNA in brain, miR-9, also preferentially localizes in neurons, and isparticularly enriched in the Purkinje cell layer (FIG. 2 e).

We also observed robust ISH signal for the less abundant miRNAs tested(FIG. 2 f-i), with unique miRNA distributions. Interestingly, miR-26aand miR-26b, which differ by 2 nucleotides (C11U, C21U) and originatefrom different clusters, showed differential expression patterns in themouse cerebellum (FIG. 2 h,i). The absolute signal intensities for themiRNAs tested were not directly correlated with the abundance of miRNAsand were likely attributed to differences in kinetics of probehybridization. Nevertheless, for a given probe, the signal intensitiesaccurately reflected miRNA differential expression.

Example 3

To examine probe hybridization specificity, we selected miR-124 andintroduced mismatches in the probe central regions. The ISH wasperformed at constant temperature 20° C. below the T_(M) of the fullycomplementary miR-124 probe. The probes with the greatest difference inT_(M) showed the least signal and central mismatches abolished detection(FIG. 15). Interestingly, signal strength observed for mismatched probeswith minor reduction in T_(M) dropped disproportionately, againindicative of altered kinetics of probe hybridization.

Example 4

Instead of a mismatch approach to control for probe specificity,conducting ISH using two probes directed against the mature miRNA andthe opposing fragment in the miRNA duplex, known as the miRNA* sequenceand/or clustered members may be useful to help rule out signals derivedfrom cross-hybridization. We probed for cistronically expressed miRNAsthat are distinct in sequence for colocalization, including members inthe mir-99a/mir-125b-1/let-7c-1 cluster. The ISH for let-7c and miR-99areveal mostly identical patterns that showed superimposable band-likepatterns (FIG. 15 g). Other let-7 family members also show similarexpression, however, the signal observed in the band-like region of thecortex likely originated from let-7c due to colocalization of miR-99a.We also tested the miRNA* sequences as a specificity control for miRNAISH signals. We examined miR-140 and its complementary miR-140*sequence, expressed at relative clone frequency of 5 to 1, respectively.The ISH with both probes revealed superimposable expression in thecortex (FIG. 15 h).

Example 5

miRNAs are known to localize to subcellular compartments for localregulation of mRNA, possibly to the dendrites of neurons. We testedseveral miRNAs for expression in dendrites including miR-370 (FIG. 3a-d), miR-9 (FIG. 3 e), and miR-124 (FIG. 3 g,h), which extended up to50 μm from the cell body. Interestingly, compared to miR-9, the 20-foldless expressed miR-9*, did not reveal localization in dendrites (FIG. 3f). Together, these data demonstrate this method detects miRNAs insubcellular compartments.

Example 6

Finally, to broaden this technique for sections not amenable tofluorescent imaging, we compared the NBT/BCIP pigment detection systemfor miR-370 in the brain to fluorescent detection and observed similarstaining (FIG. 16). We also tested the pigment detection for miR-122 andmiR-126-3p in the mouse liver, miR-1 in the mouse heart, and observedcell-type-specific staining (FIG. 17). Furthermore, we combined thedetection of proteins by immunohistochemistry with miRNA ISH (FIG. 14),although immunostaining that preceded EDC fixation reduced the signalstrength for the miRNA ISH.

Example 7

To further improve the formaldehyde-EDC-based miRNA ISH procedure, westudied the less abundant miR-129-3p (clone count frequency 0.35%) andmiR-130a (clone count frequency 0.12%), which show robust detection informaldehyde-EDC fixed tissues (FIG. 4 a, c; left), but the signalnearly blank in formaldehyde based ISH procedures (FIG. 4 b, d; left).miR-129-3p showed expression in distinct brain areas (FIG. 5 a, b) andalso expressed in the mouse hippocampus, cerebral cortex and cerebellum(FIG. 5, a-d).

Example 8

To determine if the formaldehyde-EDC miRNA ISH signal intensity is pHdependent, we treated formaldehyde fixed tissues with EDC solution in1-methylimidazole buffer at pH 5.5 and 6.0, and observed notably weakerISH signal for miR-129-3p (FIG. 6 a, b), compared to tissues fixed withEDC solution at pH 6.5 and above (FIG. 6, c-h). The most intense miRNAISH signal occurred using EDC solution at pH 7.0-8.5 (FIG. 6, c-g) andthe signal becomes moderately less intense at pH 9.0 (FIG. 6 h). Toavoid loss of signal minor fluctuations in pH, we recommend working withEDC solution at pH 8.0 (FIG. 6 f). We also examined the dose dependenceof miRNA ISH signal intensity for various EDC concentrations diluted in0.13 M imidizole buffer and pH adjusted to 8.0 (FIG. 7 a-h), and theconcentration of 0.16 M showed the brightest signal for miR-130a (FIG. 7e).

Example 9

We evaluated an alternative fixation reagent, with the goal of ligatingthe miRNA 5′ phosphate to amino groups present in the formaldehyde fixedprotein matrix, and examined the fixative cyanogen bromide (BrCN), whichforms stable phosphoramidate cross-links by condensing phosphates toamino groups {Fedorova, 1996 #70}. We applied 0.5 M BrCN in 0.16 MN-morpholinoehanesulfonate (MES)-buffer (initial pH adjusted to 8.0) toformaldehyde fixed tissues for 1 h and processed the sections for miRNAISH.

We observed a notable improvement of the miRNA ISH signal for miR-129-3pin tissues fixed with formaldehyde-EDC and formaldehyde-BrCN (FIG. 8 a,b), compared to signal from tissues fixed with formaldehyde alone (FIG.8 c).

We also examined the pH dependence for formaldehyde-BrCN solution. Intissues fixed with BrCN solutions diluted with MES-buffer at an initialpH 5.5-8.5, we observed a substantially improvement in miRNA ISH signal(FIG. 9 a-g), with the most intense signal at pH 7.0 (FIG. 9 d), andalmost no signal at pH 9.0 (FIG. 9 h). When compared to tissues fixedwith formaldehyde alone (FIG. 9 i), applying formaldehyde BrCN at pH 7.0substantially improved the signal (FIG. 9 d), and formaldehyde-BrCNsignal equaled the intensity of the formaldehyde-EDC fixed tissues (FIG.9 j).

Example 10

We examined combinations of formaldehyde, EDC, and BrCN in varioussequence order (FIG. 10 a-e), and we observed the most intense miRNA ISHsignal in tissues fixed with formaldehyde-EDC-BrCN (FIG. 10 e, EDCsolution diluted in 1-methylimidizole, pH adjusted to 8.0 and BrCNsolution diluted in MES buffer, initial pH 7.0), when compared to othercombinations (FIG. 10 a-e). Without being bound by theory, theimprovements in miRNA ISH signal intensity may be due to differingmechanisms of action in condensing the miRNA 5′ phosphate to the aminogroups in the protein matrix. In the presence of an MES-buffer, BrCNforms the condensed products considerably faster (1-3 min), compared tothe reaction rate of EDC in imidazole buffer (3-20 h), therefore theligation mechanism of action likely differ {Fedorova, 1996 #70}. Thereaction efficiency may depend on reacting groups or the adjacentnucleobases {Dolinnaya, 1994 #71}, therefore the combination of thefixatives may provide a wider coverage for miRNA which varying insequence.

The foregoing description of some specific embodiments providessufficient information that others can, by applying current knowledge,readily modify or adapt for various applications such specificembodiments without departing from the generic concept, and, therefore,such adaptations and modifications should and are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation. In the drawings and the description, there have beendisclosed exemplary embodiments and, although specific terms may havebeen employed, they are unless otherwise stated used in a generic anddescriptive sense only and not for purposes of limitation, the scope ofthe claims therefore not being so limited. Moreover, one skilled in theart will appreciate that certain steps of the methods discussed hereinmay be sequenced in alternative order or steps may be combined.Therefore, it is intended that the appended claims not be limited to theparticular embodiment disclosed herein.

1. A method for fixing a short nucleic acid in a biological sample, saidmethod comprising a) contacting the biological sample with analdehyde-containing fixative; and b) subsequently contacting the samplewith a water-soluble carbodiimide.
 2. The method of claim 1, wherein thealdehyde-containing fixative is formaldehyde.
 3. The method of claim 1,wherein the carbodiimide is selected from the group consisting of:1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC);1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), andN,N′-diisopropylcarbodiimide (DIC).
 4. The method of claim 3, whereinthe carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. 5.The method of claim 4, wherein the1-ethyl-3-(3-dimethylaminopropyl)carbodiimide has a concentration ofabout 50 mM to about 250 mM.
 6. The method of claim 4, wherein the1-ethyl-3-(3-dimethylaminopropyl)carbodiimide contacts the sample at atemperature of about 20° C. to about 70° C.
 7. The method of claim 4,wherein the carbodiimide has a pH of 8.0 in solution.
 8. A method fordetecting a target short nucleic acid in a biological sample, saidmethod comprising a) contacting the biological sample with analdehyde-containing fixative; b) subsequently contacting the sample witha water-soluble carbodiimide to produce a crosslinked short nucleicacid; c) contacting the cross-linked miRNA with a probe, said probebeing complementary to all or a part of a region of interest of theshort nucleic acid, thereby producing a hybridized short nucleic acid;and d) detecting the hybridized short nucleic acid as the target shortnucleic acid.
 9. The method of claim 8, wherein the carbodiimide isselected from the group consisting of:1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC);1-Cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimidemetho-p-toluenesulfonate (CMC), N,N′-dicyclohexylcarbodiimide (DCC), andN,N′-diisopropylcarbodiimide (DIC).
 10. The method according to claim 9,wherein the agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. 11.The method according to claim 10, wherein the1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has a concentration ofabout 50 mM to about 250 mM.
 12. The method according to claim 10,wherein the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide has atemperature of about 20° C. to about 70° C.
 13. The method according toclaim 8, wherein the probe comprises a locked nucleic acid residue. 14.A kit for fixing a short nucleic acid in a biological sample, comprisinga) a support substrate for holding the sample; b) an aldehyde-containingfixative; and c) a water-soluble carbodiimide.
 15. The kit according toclaim 14, wherein the aldehyde-containing fixative is formaldehyde. 16.The kit of claim 14, wherein the agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.